Central nervous system (CNS) injuries to are a serious health problem. This category of injury includes events such as ischemic injury, hemorrhagic injury, penetrating trauma and non-penetrating trauma. CNS injuries generally heal incompletely leaving the subject with some degree of permanent dysfunction ranging from extremely mild to death. The residual dysfunction may include motor, sensory, cognitive, emotional and autonomic abnormalities.
A key category of CNS neuroinjury comprises brain injury. Brain injury is a devastating condition that results in some degree of permanent disability including motor, sensory and cognitive deficits and emotional instability such as post traumatic stress disorder, attention deficit disorder, depression and emotional lability. Common causes of brain injury include ischemic stroke, hemorrhagic stroke, subdural hematoma, epidural hematoma, closed head injury (acceleration/deceleration, concussion and rotational), penetrating brain injury (gunshot wounds and other projectiles).
Stroke is the third-leading cause of death and the main cause of disability in the western world. Stroke, therefore, presents a large socioeconomic burden. The etiology of a stroke can be either ischemic, which is the case in the majority of strokes, or hemorrhagic. An ischemic stroke can be caused by a clot that forms elsewhere in the body and travels via the bloodstream to the brain (embolic stroke) or by a blood clot that forms inside the artery of the brain (thrombotic stroke). After massive cell death in the immediate infarct core due to lack of glucose and oxygen, the infarct area expands for days, owing to secondary mechanisms such as glutamate excitotoxicity, apoptotic mechanisms, and generation of free radicals. Following neuroinjuries (e.g. an ischemic event) animals and people may recover function over several days, weeks and months without any therapeutic. All too often, this recovery however is only partial and animals and people suffer from permanent disability which may include motor, sensory and cognitive deficits.
Risk factors that increase the likelihood of an individual having a stroke are well known. These include, and are not limited to, risk factors that cannot be changed: advanced age, heredity, ace, gender, prior history of stroke or heart attack; and risk factors that can be changed, treated or controlled: high blood pressure, cigarette smoking, diabetes mellitus, carotid or other artery disease, atrial fibrillation, other heart disease, sickle cell disease, high blood cholesterol, poor diet, and physical inactivity and obesity.
To date, the non-palliative treatment of ischemic stroke is confined to therapeutics administered in the acute phase following a stroke. The acute phase ranges from the time of onset of the neuroinjury (e.g., stroke) to approximately six hours post-neuroinjury. The acute phase is followed by the semi-acute phase, which ranges from approximately six hours to two days post-neuroinjury. Accordingly, current non-palliative therapeutics are used in an attempt to reverse the occlusion of blood flow, restore oxygenation of the brain and limit the extent of lost brain structure. Other than tPA for acute use, there are no drugs are approved for the treatment of stroke. Patients remain with some level of dysfunction that at best may improve somewhat endogenously for approximately 60 days. This recovery may only be augmented by physical therapy. Unfortunately, many patients are left with permanent disability with little hope for improvement.
At present, the only drug approved by the Food and Drug Administration (FDA) for the treatment of ischemic stroke is tissue plasminogen activator (tPA). tPA is a serine protease that converts plasminogen to plasmin. Plasmin then breaks fibrin which is a component of the clots that occlude the vessels in the brain and cause strokes. It is ideally administered within three hours from the onset of symptoms. Generally, only 3% to 5% of individuals who suffer a stroke reach the hospital in time to be considered for this treatment. Ideally, tPA is administered within the first three hours post-occlusion, but may be administered by some clinicians as late as six hours post-occlusion. Unfortunately, the vast majority of patients who experience a stroke fail to reach the hospital in time to be considered for this treatment. For those patients who arrive at the hospital within the efficacious temporal window, tPA is administered in an attempt to reverse the occlusion of blood flow, restore oxygenation of the brain and limit the extent of lost brain structure. However, there are some significant contraindications that limit the ongoing use of tPA. After an initial period of about 3 to 6 hours, at most, tPA can cause intracerbral bleeding and hemorrhagic stroke. For such reasons, tPA is limited to administration during the acute phase in order to achieve any therapeutic efficacy.
To date no other therapy has been approved for the treatment of stroke. Other experimental therapies such as arterially delivered pro-urokinase are under investigation as potential means for disrupting clots and restoring blood flow. The scientific literature has, however, described many agents that have proven beneficial for protecting brain matter and restoring function in experimental animal models of stroke. All these agents focus on reducing acute cell death, inflammation, and apoptosis and must, therefore, be delivered within hours (some up to 24 hours) after the ischemic event. Heretofore, it is generally accepted that treatment for CNS injury such as stroke is required acutely. (Abe et al., 2008, J Cereb Blood Flow Metab. July 23, Epub ahead of print, Sun et al., 2008, Stroke July 10, Epub ahead of print, pages not available yet); Dohare et al., 2008, Behav Brain Res. 193(2):289-97; Belayev et al., 2001, Stroke 32(2):553-60).
Such agents have not, however, been shown to limit damage to the brain, restore function or enhance recovery following stroke when administered after a lag time of several hours, at most in some experimental animal models about one day following stroke. The only therapy known to show efficacy days and weeks after a stroke is palliative or rehabilitative, such as occupational or physical therapy. Indeed, the present inventors are not aware of any agents or drugs that have been shown to enhance recovery days or weeks following stroke.
After an acute occlusion, there is often a localized area of destroyed brain matter that is surrounded by a penumbral zone that will die within hours if circulation is not restored. The time to death of this penumbral zone can be extended by a few hours in experimental models with neuroprotectants, such as NMDA antagonists, calcium channel blockers, radical scavengers and trapping agents, anti-apoptotics, caspase inhibitors, parp inhibitors, etc. For this purpose a “neuroprotectant” is something that can save neurons before they die from the variety of insults presented to them in the acute phase. After 24 to 48 hours, however, there is little hope for protecting cells from necrotic death and, while apoptotic death continues for several days (See FIG. 1 the therapeutic window for anti-apoptotic therapies has not proven to be much wider than acute protective therapies [Schulz et al., 1998, Cell Death Differ. 5(10):847-57; Komjati et al., 2004, Int J Mol Med. 13(3):373-82].
Neuregulin exhibits neuroprotective properties which, like other agents described above, have shown benefit in reducing the disability seen if delivered to animals within hours after stroke. See U.S. application Ser. No. 09/530,884, the entire contents of which are incorporated herein by reference.
In view of the prevalence of neuroinjury, particularly with regard to stroke, there is a need for therapeutic agents that can be administered efficaciously to subjects to limit damage to the brain, restore function and/or enhance recovery following neuroinjury.
Neuregulins (NRGs) and NRG receptors comprise a growth factor-receptor tyrosine kinase system for cell-cell signaling that is involved in organogenesis in nerve, muscle, epithelia, and other tissues (Lemke, Mol. Cell. Neurosci. 7:247-262, 1996 and Burden et al., Neuron 18:847-855, 1997). The NRG family consists of four genes that encode numerous ligands containing epidermal growth factor (EGF)-like, immunoglobulin (Ig), and other recognizable domains. Numerous secreted and membrane-attached isoforms function as ligands in this signaling system. The receptors for NRG ligands are all members of the EGF receptor (EGFR) family, and include EGFR (or ErbB1), ErbB2, ErbB3, and ErbB4, also known as HER1 through HER4, respectively, in humans (Meyer et al., Development 124:3575-3586, 1997; Orr-Urtreger et al., Proc. Natl. Acad. Sci. USA 90: 1867-71, 1993; Marchionni et al., Nature 362:312-8, 1993; Chen et al., J. Comp. Neurol. 349:389-400, 1994; Corfas et al., Neuron 14:103115, 1995; Meyer et al., Proc. Natl. Acad. Sci. USA 91:1064-1068, 1994; and Pinkas-Kramarski et al., Oncogene 15:2803-2815, 1997).
The four NRG genes, NRG-1, NRG-2, NRG-3, and NRG-4, map to distinct chromosomal loci (Pinkas-Kramarski et al., Proc. Natl. Acad. Sci. USA 91:9387-91, 1994; Carraway et al., Nature 387:512-516, 1997; Chang et al., Nature 387:509-511, 1997; and Zhang et al., Proc. Natl. Acad. Sci. USA 94:9562-9567, 1997), and collectively encode a diverse array of NRG proteins. The gene products of NRG-1, for example, comprise a group of approximately 15 distinct structurally-related isoforms (Lemke, Mol. Cell. Neurosci. 7:247-262, 1996 and Peles and Yarden, BioEssays 15:815-824, 1993). The first-identified isoforms of NRG-1 included Neu Differentiation Factor (NDF; Peles et al., Cell 69, 205-216, 1992 and Wen et al., Cell 69, 559572, 1992), heregulin (HRG; Holmes et al., Science 256:1205-1210, 1992), Acetylcholine Receptor Inducing Activity (ARIA; Falls et al., Cell 72:801-815, 1993), and the glial growth factors GGF1, GGF2, and GGF3 (Marchionni et al. Nature 362:312-8, 1993).
The NRG-2 gene was identified by homology cloning (Chang et al., Nature 387:509-512, 1997; Carraway et al., Nature 387:512-516, 1997; and Higashiyama et al., J. Biochem. 122:675-680, 1997) and through genomic approaches (Busfield et al., Mol. Cell. Biol. 17:4007-4014, 1997). NRG-2 cDNAs are also known as Neural- and Thymus-Derived Activator of ErbB Kinases (NTAK; Genbank Accession No. AB005060), Divergent of Neuregulin (Don-1), and Cerebellum-Derived Growth Factor (CDGF; PCT application WO 97/09425). Experimental evidence shows that cells expressing ErbB4 or the ErbB2/ErbB4 combination are likely to show a particularly robust response to NRG-2 (Pinkas-Kramarski et al., Mol. Cell. Biol. 18:6090-6101, 1998). The NRG-3 gene product (Zhang et al., supra) is also known to bind and activate ErbB4 receptors (Hijazi et al., Int. J. Oncol. 13:1061-1067, 1998).
An EGF-like domain is present at the core of all forms of NRGs, and is required for binding and activating ErbB receptors. Deduced amino acid sequences of the EGF-like domains encoded in the three genes are approximately 30-40% identical (pairwise comparisons). Further, there appear to be at least two sub-forms of EGF-like domains in NRG-1 and NRG-2, which may confer different bioactivities and tissue-specific potencies
Cellular responses to NRGs are mediated through the NRG receptor tyrosine kinases EGFR, ErbB2, ErbB3, and ErbB4 of the epidermal growth factor receptor family. High-affinity binding of all NRGs is mediated principally via either ErbB3 or ErbB4. Binding of NRG ligands leads to dimerization with other ErbB subunits and transactivation by phosphorylation on specific tyrosine residues. In certain experimental settings, nearly all combinations of ErbB receptors appear to be capable of forming dimers in response to the binding of NRG-1 isoforms. However, it appears that ErbB2 is a preferred dimerization partner that may play an important role in stabilizing the ligand-receptor complex. ErbB2 does not bind ligand on its own, but must be heterologously paired with one of the other receptor subtypes. ErbB3 does not possess tyrosine kinase activity, but is a target for phosphorylation by the other receptors. Expression of NRG-1, ErbB2, and ErbB4 is known to be necessary for trabeculation of the ventricular myocardium during mouse development.